Current ceramic processing and engineering are based on a well-established sequence of processes enabling the production of large 3D bodies. More specifically, innovative ceramic phases can be synthesized as powders, where specific features such stoichiometry/ion substitutions, nanosize, and surface activity, are responsible for specific functionalities. The ceramic processing currently used to obtain macroscopic 3D ceramic bodies with adequate shape and porosity implies thermal treatment (sintering) of the synthesized ceramic powders suitably formed into a 3-D body (to consolidate the body). All these steps are needed to obtain 3D ceramics with adequate physicochemical and mechanical properties, most of which are degraded during the above-mentioned ceramic process (particularly the sintering treatment). The serious limitations in the development of functional ceramic material, associated with the current ceramic process, impede further progress in the field.
Nowadays, with the evolution of modern society, technological products are assuming a steadily increasing role in the life and productivity of people, so that there is a strong need for smart tools able to provide solutions to complex and personalized demands, in various fields of application, e.g. health, environment, energy. Therefore, there is a wide consensus that new approaches are needed for the repeatable and massive production of macroscopic devices with complex structural organization at the macro-scale but, at the same time with a complex structure defined at the nanoscale, and even at the crystal scale. Such macro and nano-structures are relevant to induce non-trivial, but smart functional effects.
With respect to the above-mentioned issues regarding ceramic materials, a paradigmatic change is required in order to develop large highly active ceramics with complex micro and macro-structures.
Bone scaffolds, with particular focus to the regeneration of large, load-bearing bone defects, can be taken as a representative example since they should be porous 3-D ceramics with high bioactivity, in order to be able to be colonized by cells and ultimately regenerated as large bone defects. Indeed, no adequate solutions have been found to date to solve this clinical need.
For many decades, hydroxyapatite (Ca10(PO4)6(OH)2) has been recognized as the prime material for bone scaffolding, as it closely resembles the composition of bone mineral and has demonstrated excellent biocompatibility and osteoconductivity. However, the biomimicry of hydroxyapatite is related to its nanosize and the presence of multiple ions, partially replacing calcium and phosphate in the apatite lattice, which are the source of the biological activity of the bone during new tissue formation, remodeling and resorption.
The application of the sintering treatment to hydroxyapatite scaffolds activates surface and bulk reactions at the interface between adjacent hydroxyapatite grains that yield crystal ordering, with expulsion of foreign ions from the apatite lattice, and grain coalescence up to several micrometers, with reduction of specific surface, hydrophilicity and affinity with proteins and cells.
The extensive grain coalescence activated by the sintering process yields consolidation of the whole hydroxyapatite body through reduction of the intergranular porosity and, in turn, of the overall volume. This also generates residual stresses which are among the main sources of structural defects in the ceramic materials. Indeed, the accommodation of residual stresses in ceramic materials is difficult due to their high rigidity (compared to metals and polymers), and is among the most significant factors impairing the mechanical performance of ceramic materials, particularly in the case of large pieces characterized by complex shapes and porous structures, where volume variations following heating/cooling cycles easily provoke critical structural damage.
For the above reasons, the classical ceramic synthesis process does not allow ceramic materials, in particular hydroxyapatites having a biomimetic composition and structure, high bioactivity and resorbability, to be manufactured. This is especially true when large porous 3D ceramics are synthesized for the regeneration of critical size bone defects (i.e. ≥2 cm).
Biomimetic composition and structure are of pivotal relevance for inducing the regenerative cascade in vivo that can uniquely determine and promote regeneration of large, load-bearing bone parts such as the long bones of the limbs. These phenomena, which are closely inter-related and must occur in synergy to activate and sustain the regeneration of bone with all its functions, are: i) fast osteogenesis, osteoconduction and osteointegration; ii) extensive blood vessel formation; iii) ability of progressive bio-resorption.
Fast osteogenesis and osteoconduction enable extensive bone formation and penetration into the scaffold, thus resulting in tight bone/scaffold interface and optimal osteointegration. To achieve these effects, bone-like chemical composition as well as wide open and interconnected porosity are required, so that besides extensive penetration of new bone tissue, a simultaneous formation of a vascular network assisting the formation and maturation of the new bone can be achieved. Incomplete colonization of the scaffold may result in the formation of voids, fibrous tissues or necrotic areas, and will reduce the overall strength and biomechanical performance of the bone/scaffold construct.
Within times compatible with new bone formation, the scaffold should be progressively resorbed, to achieve optimal regeneration of the bone following damage or disease. All the 3D bone scaffolds developed so far are based on sintered calcium phosphates that are crystalline materials hampering osteoclast activity, compared to nanocrystalline, nanosized, ion-substituted apatite; therefore, even though porous bone hydroxyapatite scaffolds can be well integrated into the surrounding bone by surface adhesion, the lack of bio-resorption does not allow the complete remodeling process, i.e. replacement of the scaffold with the new bone. This results in incomplete recovery of the functional ability of the diseased bone, particularly in the case of very long, load-bearing, bone segments.
Particularly in the case of long, load-bearing bones, the scaffold must also exhibit adequate mechanical performance, while maintaining wide open macro-porosity, which is a challenge considering that these features are normally inversely related (i.e. the higher the porosity, the lower the mechanical resistance) and that a high porosity extent is required to provide adequate scaffold colonization and osteointegration. This is one of the most relevant factors limiting the application of current scaffolds in the regeneration of extensive portions of long, load-bearing bones. In this respect, scaffolds with hierarchically-organized porous structures can exhibit superior mechanical performance compared to materials with similar, but randomly organized porosity. In this respect, only scaffolds with such an organized structure can efficiently activate mechano-transduction processes at the cell level, thus triggering regeneration of mature, organized and mechanically-competent bone.
The proposed innovation is based on a paradigmatic change from the classical ceramic synthesis process to a new fashion of reactive sintering that enables the generation of ceramic phases with defined chemical composition, organized into a large 3D body with complex morphology, hierarchical structure and, at the same time, optimized mechanical performance, starting from hierarchically organized natural structures. In this respect biomorphic transformation is the fulcrum of this innovative approach that can be applied to hierarchically organized natural structures (e.g. woods, plants, exoskeletons).
Biomorphic transformation of ligneous structures to bone-mimicking ceramics was successfully attempted using woods with porous structures such as pinewood and rattan, and denser woods such as red oak and sipo, as templates for reproducing the structure and mechanical performance of spongy and cortical bone, respectively. The use of wood in the formation of biomimetic hydroxyapatite scaffolds was reported by Anna Tampieri et al. in the Journal of Material Chemistry, 2009, 19, 4973-4980. In this publication, Tampieri et al. describe the process of converting 1 cm long pieces (therefore a small piece, not adequate for regeneration of critical size defects) of rattan wood and pine wood into hydroxyapatite. The process involved pyrolysis of the wood specimens at a temperature of 1000° C. using a slow heating rate, followed by carburization wherein the carbon template was transformed into calcium carbide. Carburization was achieved by either liquid phase infiltration or vapour phase infiltration. Vapour infiltration was performed at temperatures higher than the boiling point of calcium (1484° C.). The carburization process involved initial heating the pyrolised wood to 800° C., followed by heating to 1100° C. and finally to 1650° C. for 3 hours. It was necessary to heat the pyrolised wood to this temperature for 3 hours to ensure that the reaction went to completion. Following carburization, the three dimensional calcium carbide scaffold was oxidized to transform the calcium carbide to calcium oxide, while preserving the morphology of the native wood. After oxidation, the three dimensional calcium oxide scaffold was carbonated to transform the calcium oxide scaffold into calcium carbonate scaffold. High pressure values (2.2 MPa) were employed to allow the penetration of CO2 across the forming CaCO3 scale, up to the core of the CaO structure. Finally, a phosphatization step was carried out to transform the calcium carbonate scaffold into hydroxyapatite scaffold with hierarchically organized anisotropic morphology resembling that of the native wood. During this step, the wood-derived CaCO3 templates were soaked in an aqueous solution of KH2PO4 at a temperature of 200° C., under a pressure of 1.2 MPa for 24 hours.
The process described above yielded hydroxyapatite ceramic scaffolds with the hierarchically organized anisotropic morphology of native wood.
The compressive strength of the scaffold derived from pinewood, measured in the longitudinal direction ranged between 2.5 and 4 MPa, and in the transversal direction, ranged between 0.5 and 1 MPa. Therefore only scaffolds of limited dimension, typically of less than 1 cm, are obtainable by said process. The low values of compression strength, also in association with a size ≤1 cm, make these scaffolds not relevant for bone regeneration, particularly in the case of load-bearing bones. In fact, it is accepted that, to be critical, a bone defect should have a length of 2-3 times the diameter of the affected bone. Hence, scaffold of 1 cm in size cannot be considered as useful in this respect.
The phosphatization step mentioned above in the conversion of wood to hydroxyapatite, was reported in more detail by Ruffini et al. in Chemical Engineering Journal 217 (2013) 150-158. In this publication, cylindrical templates of rattan-derived calcium carbonate having diameters of 8 mm and lengths of 10 mm were used as starting materials. The phosphatization process was carried out using aqueous solutions of diammonium hydrogen phosphate, ammonium dihydrogen phosphate and ammonia.
Patent application WO 2012/063201 published on 18 May 2012, describes a bone substitute comprising a core, based on hydroxyapatite, obtained from at least one porous wood, and a shell based on hydroxyapatite or silicon carbide obtained from at least one wood having a lower porosity than the at least one wood of the core. The shell was prepared in a hollow cylindrical shape suitable for accommodating the core, which could be prepared as a solid cylinder that is inserted into the cavity of the shell. The process for obtaining the bone substitute from wood is also described in the application. The first step is pyrolysis of a native wood such as rattan or pine, by heating it to a temperature of between 800 and 2000° C. From this process, a carbon material is obtained. In the second step, the carbon material is transformed into calcium carbide at a temperature of between 1500 to 1700° C. Next, the calcium carbide is oxidized at a temperature between 900 and 1000° C. In order to convert the calcium oxide material to calcium carbonate, carbonation is performed in an autoclave at a temperature of 400° C. with a CO2 pressure of 2.2 MPa for 24 hours. The calcium carbonate material is then transformed into hydroxyapatite partially substituted with carbonate by phosphatization. The resulting hydroxyapatite scaffolds derived from rattan, have a compressive strength of between 4 and 5 MPa in the longitudinal direction, and a compressive strength of 1 MPa in the transversal direction.
Although the publications mentioned above describe the successful transformation of wood such as rattan and pine into hydroxyapatite, while fairly reproducing the three-dimensional morphology of the wood, scaffolds exhibiting features adequate for regeneration of long segments of load-bearing bone could not be obtained.
Indeed all of the mentioned publications refer to hydroxyapatite scaffolds obtained from wood, having small dimensions (i.e. a volume of less than 1 cm3) that cannot have real clinical applications, particularly for the regeneration of large, load-bearing bone parts. The processes described in the prior art are not suitable for manufacturing hydroxyapatite scaffolds having dimensions that are convenient for clinical applications, such as for the regeneration of critical size load-bearing bone defects where large scaffolds, i.e. with size at least equal to 2 cm, are needed.
Thus there remains a need in the art for a biomorphic scaffold, in particular a porous 3D scaffold, with a biomimetic chemical composition that exhibits adequate mechanical performance, a morphology that is favorable to cell colonization and vascular growth and, at the same time, which has dimensions that are suitable for clinical applications.
Such a biomorphic scaffold would be particularly suitable for bone regeneration, in particular for implantation in load-bearing bone defects, such as long bones of the limbs (e.g. femur, tibia, humerus, fibula, radius), but also for the substitution and regeneration of spine bones (e.g. vertebral bodies, intervertebral disc), cranial bone-parts or maxillofacial bone-parts.
The present disclosure meets the above needs by providing a biomorphic scaffold, preferably a hydroxyapatite scaffold particularly suitable for bone substitution and regeneration, in particular for substitution and regeneration of long load-bearing bones.
The present disclosure meets the above needs also by providing a process for the manufacturing of a biomorphic scaffold, preferably a 3D biomorphic scaffold. In particular, the biomorphic scaffold is a hydroxyapatite scaffold.